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| 2. |
- Fornander, Louise, 1984, et al.
(författare)
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Using nanofluidic channels to probe dynamics of RAD51-Filaments
- 2015
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Ingår i: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014. - 9780979806476 ; , s. 1826-1828
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Konferensbidrag (refereegranskat)abstract
- Using nanochannels, passivated with a lipid bilayer to avoid sticking of proteins, we study Rad51 filaments bound to single- and double stranded DNA. We demonstrate how we can discern different properties of the filaments by studying them at different degrees of confinement. Unlike the bacterial homologue RecA, that forms homogeneous filaments along DNA, Rad51 forms heterogeneous filaments containing both rigid kinks as well as flexible regions. Varying the counterion, the DNA substrate as well as the initial protein concentration, we try to understand the factors governing the structure of the filaments.
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| 3. |
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| 4. |
- Fornander, Louise, 1984, et al.
(författare)
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Visualizing the Nonhomogeneous Structure of RAD51 Filaments Using Nanofluidic Channels
- 2016
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Ingår i: Langmuir. - : American Chemical Society (ACS). - 0743-7463 .- 1520-5827. ; 32:33, s. 8403-8412
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Tidskriftsartikel (refereegranskat)abstract
- RAD51 is the key component of the homologous recombination pathway in eukaryotic cells and performs its task by forming filaments on DNA. In this study we investigate the physical properties of RAD51 filaments formed on DNA using nanofluidic channels and fluorescence microscopy. Contrary to the bacterial ortholog RecA, RAD51 forms inhomogeneous filaments on long DNA in vitro, consisting of several protein patches. We demonstrate that a permanent "kink" in the filament is formed where two patches meet if the stretch of naked DNA between the patches is short. The kinks are readily seen in the present microscopy approach but would be hard to identify using conventional single DNA molecule techniques where the DNA is more stretched. We also demonstrate that protein patches separated by longer stretches of bare DNA roll up on each other and this is visualized as transiently overlapping filaments. RAD51 filaments can be formed at several different conditions, varying the cation (Mg2+ or Ca2+), the DNA substrate (single-stranded or double-stranded), and the RAD51 concentration during filament nucleation, and we compare the properties of the different filaments formed. The results provide important information regarding the physical properties of RAD51 filaments but also demonstrate that nanofluidic channels are perfectly suited to study protein-DNA complexes.
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| 5. |
- Frykholm, Karolin, 1977, et al.
(författare)
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Fast size-determination of intact bacterial plasmids using nanofluidic channels
- 2015
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Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 15:13, s. 2739-2743
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Tidskriftsartikel (refereegranskat)abstract
- We demonstrate how nanofluidic channels can be used as a tool to rapidly determine the number and sizes of plasmids in bacterial isolates. Each step can be automated at low cost, opening up opportunities for general use in microbiology labs.
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| 6. |
- Frykholm, Karolin, 1977, et al.
(författare)
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Probing Physical Properties of a DNA- Protein Complex Using Nanofluidic Channels
- 2014
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Ingår i: Small. - : Wiley. - 1613-6810 .- 1613-6829. ; 10:5, s. 884-887
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Tidskriftsartikel (refereegranskat)abstract
- A method to investigate physical properties of a DNA-protein complex in solution is demonstrated. By using tapered nanochannels and lipid passivation the persistence length of a RecA filament formed on double-stranded DNA is determined to 1.15 μm, in agreement with the literature, without attaching protein or DNA to any handles or surfaces.
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| 7. |
- Frykholm, Karolin, 1977, et al.
(författare)
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Probing physical properties of DNA-protein complexes using nanofluidic channels
- 2013
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Ingår i: 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013; Freiburg; Germany; 27 October 2013 through 31 October 2013. - 9781632666246 ; 2, s. 1311-1313
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Konferensbidrag (refereegranskat)abstract
- We present the use of nanofluidic channels as a tool for determining physical properties of single DNA-protein complexes. By coating the nanochannels with a lipid bilayer we avoid sticking of proteins to the channel walls. RecA is a prokaryotic protein involved in recombination and DNA repair. We study filaments of RecA, bound to both double stranded (ds) and single stranded (ss) DNA. We determine the persistence length of RecA filaments on both dsDNA and ssDNA and obtain values in agreement with the literature. Neither the DNA nor the protein has to be attached to handles or surfaces, and the technique is directly transferable to Lab-on-a-Chip technologies for high throughput measurements in solution.
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| 8. |
- Müller, Vilhelm, 1990, et al.
(författare)
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Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping.
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 6, s. 37938-
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples.
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| 9. |
- Werner, Erik, et al.
(författare)
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Hairpins in the conformations of a confined polymer
- 2018
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Ingår i: Biomicrofluidics. - : AIP Publishing. - 1932-1058. ; 12:2
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Tidskriftsartikel (refereegranskat)abstract
- If a semiflexible polymer confined to a narrow channel bends around by 180°, the polymer is said to exhibit a hairpin. The equilibrium extension statistics of the confined polymer are well understood when hairpins are vanishingly rare or when they are plentiful. Here, we analyze the extension statistics in the intermediate situation via experiments with DNA coated by the protein RecA, which enhances the stiffness of the DNA molecule by approximately one order of magnitude. We find that the extension distribution is highly non-Gaussian, in good agreement with Monte-Carlo simulations of confined discrete wormlike chains. We develop a simple model that qualitatively explains the form of the extension distribution. The model shows that the tail of the distribution at short extensions is determined by conformations with one hairpin. © 2018 Author(s).
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| 10. |
- Dvirnas, Albertas, et al.
(författare)
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Detection of structural variations in densely-labelled optical DNA barcodes: A hidden Markov model approach
- 2021
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 16:11
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Tidskriftsartikel (refereegranskat)abstract
- Large-scale genomic alterations play an important role in disease, gene expression, and chromosome evolution. Optical DNA mapping (ODM), commonly categorized into sparsely-labelled ODM and densely-labelled ODM, provides sequence-specific continuous intensity profiles (DNA barcodes) along single DNA molecules and is a technique well-suited for detecting such alterations. For sparsely-labelled barcodes, the possibility to detect large genomic alterations has been investigated extensively, while densely-labelled barcodes have not received as much attention. In this work, we introduce HMMSV, a hidden Markov model (HMM) based algorithm for detecting structural variations (SVs) directly in densely-labelled barcodes without access to sequence information. We evaluate our approach using simulated data-sets with 5 different types of SVs, and combinations thereof, and demonstrate that the method reaches a true positive rate greater than 80% for randomly generated barcodes with single variations of size 25 kilobases (kb). Increasing the length of the SV further leads to larger true positive rates. For a real data-set with experimental barcodes on bacterial plasmids, we successfully detect matching barcode pairs and SVs without any particular assumption of the types of SVs present. Instead, our method effectively goes through all possible combinations of SVs. Since ODM works on length scales typically not reachable with other techniques, our methodology is a promising tool for identifying arbitrary combinations of genomic alterations.
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